One aspect of this project involves exploring the importance of the biosynthesis of various cofactors, cell wall assembly and turnover, and transcriptional and translational systems that maintain chromosomal integrity and replication in non-replicating (NR)-MTb. For example, we have previously demonstrated that NAD recycling and biosynthesis was critical for viability of MTb under both active replicating conditions as well as non-replicating persistence by genetic and chemical genetic methods. We have now used conditional expression mutants of the essential nadE gene which encodes NAD synthetase to show that depletion of this protein results in rapid bacterial cell death in both actively growing as well as NR bacteria in vitro and also during acute and chronic TB infection in mice. We are continuing our systematic analysis of potential bottlenecks in the coenzyme A metabolic pathway to identify potential high-value drug targets. Conditional expression systems for each enzymatic step have been generated to identify those steps in the pathway that are most sensitive to inhibition. These chokepoints in coenzyme A biosynthesis are potentially the most sensitive to chemical inhibition and represent the best drug targets. The major secreted protein antigens of MTb are a family of three closely-related proteins that have clear enzymatic function in constructing the unique cell wall of this organism. Despite knowledge of the reaction they catalyzed the reason for the existence of three, apparently redundant isoforms has never been understood. We have fully characterized the three isoforms of Antigen 85 enzymologically and noted the presence of adventitious ligands in a non-active site binding pocket. Using site-directed mutagenesis we showed that this secondary binding site confers substrate specificity and overall activity of the enzymes when occupied by carbohydrate ligands. By creating chimeric proteins we were able to define the unique functions of each of these proteins and provide a framework for developing potent inhibitors. Drug resistance in MTb is most commonly seen as a result of alterations in the binding site of a drug and its target. These mutations often have deleterious effects on the normal function of the mutated protein and organisms containing these mutant proteins often have significant growth impairment, an observation that is often used to argue that drug resistant TB presents only a relatively small threat. We studied the major mutations in the TB RNA polymerase that gave rise to rifpampicin resistance in a large collection of hundreds of highly drug resistant isolates from South Korea and through whole genome sequence analysis identified a series of second-site mutations that were acquired in some MDR and XDR isolates in unique patterns. The binding site and rifampicin resistance causing mutations were in the Beta subunit of the polymerase but we observed second site mutations relatively distant to these in the beta' subunit. Some of these occurred in discrete clusters of highly related organisms suggesting an underlying outbreak of a single clone but other pairs of mutations occurred convergently in epidemiologically unlinked isolates suggesting these were the result of strong selective pressure. We created a laboratory strain that contained the predominant mutation in the RNA polymerase that confers Rifampicin resistance in these isolates and showed that this resulted in an organism that was severely attenuated for growth under specific conditions and that this growth restriction was fully restored by the clinically observed compensatory mutations. We subsequently retrospectively re-analyzed genome sequences from the Western Cape in South Africa and noted the same combination had also emerged in XDR isolates from this region. Together these findings suggest that evolution of some highly drug-resistant strains of TB has already progressed to the point where rifampicin resistance is permanently and stably fixed in these populations. Similarly we have also analyzed the effects of two related inhibitors of the folate pathway, para-aminosalicylic acid (PAS) and fenamisal, and found that although resistant mutants to either tend to overlap in terms of cross-resistance, the spectrum of mutations elicited by the individual drugs are different. In addition, metabolomics analyses showed that effects on pools of metabolites directly related to folate metabolism were distinct. Biochemical analyses showed that both PAS and fenamisal functioned as anti-metabolites by being incorporated into the folate pathway but forming functionally distinct analogs. Sequencing of clinical strains with resistance that had emerged in patients due to chemotherapy with PAS, showed that the majority of mutations were not in the target but in a folate consuming enzyme and that compensatory mutations emerged that likely generated strains with higher fitness in the human lung environment.